Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The mortality rate is roughly that of cancer. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis.[unreadable] The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3-5 exonuclease proofreading activity, and 5dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are nearly 100 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO).[unreadable] PEO is a mitochondrial disorder associated with mtDNA deletions and point mutations. PEO is characterized by late onset (between 18 and 40 years of age) bilateral ptosis and progressive weakening of the external eye muscle, resulting in blepharoptosis and ophthalmoparesis, proximal muscle weakness and wasting as well as exercise intolerance. The disease is often accompanied by cataract, hypogonadism, dysphagia, hearing loss and may, within several years, lead to development of neuromuscular problems. Neurological problems may include depression or avoidant personality. Skeletal muscles of PEO patients present red ragged fibers and lowered activity of respiratory chain enzymes. PEO can be inherited in an autosomal dominant or recessive manner.[unreadable] Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder that affects children and young adults. It is a devastating disease characterized by psychomotor retardation, hepatic failure and intractable seizures, as well as tissue-specific mtDNA depletion. Alpers syndrome is 100% fatal with no cure available. This syndrome has been exclusively associated with mutations in POLG. Carriers for this disease are 1:100 with presentation occurring 1:10,000. We and others have identified over 40 pathogenic POLG mutations that cause Alpers in over 50 probands.[unreadable] Ataxia/neuropathy resulting from mutations in POLG is an autosomal recessive disorder affecting patients in their mid-teens to later years usually resulting in premature death. The disease is accompanied mainly by mtDNA deletions. The ataxia usually occurs in combination with various central nervous system features including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and evidence of neuronal loss in discrete gray nuclei.[unreadable] Presently, there are nearly 100 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome.[unreadable] The W748S mutation in POLG is the most common mutation in ataxia-neuropathy spectrum disorders and is generally found in cis with the common E1143G polymorphism. It has been unclear whether E1143G participates in the disease process. We investigated the biochemical consequences of pol gamma proteins containing W748S or E1143G, or both. W748S pol gamma exhibited low DNA polymerase activity, low processivity and a severe DNA-binding defect. However, interactions between the catalytic and accessory subunits were normal. Despite the benefits derived from binding with the accessory subunit, catalytic activities did not reach wild-type (WT) levels. Also, nucleotide selectivity decreased 2.1-fold compared with WT. Surprisingly, pol gamma containing only E1143G was 1.4-fold more active than WT, and this increased polymerase activity could be due to higher thermal stability for E1143G pol gamma. The E1143G substitution partially rescued the deleterious effects of the W748S mutation, as DNA binding, catalytic activity and fidelity values were intermediate for W748S-E1143G. However, W748S-E1143G had a notably lower change in enthalpy for protein folding than W748S alone. We suggest that when E1143G is in cis with other pathogenic mutations, it can modulate the effects of these mutations. For W748S-E1143G pol gamma, the benefits bestowed by E1143G include increased DNA binding and polymerase activity; however, E1143G was somewhat detrimental to protein stability.[unreadable] We have developed an animal model of a POLG mitochondrial disease by expressing the human POLG cDNA with the Y955C mutation in a mouse transgenic model where the human Y955C pol gamma is targeted to the mouse hearts. The Y955C mutation is associated in humans with autosomal dominant progressive external ophthalmoplegia and parkinsonism. Survival of the mouse transgene was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy were all investigated. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.[unreadable] In the human DNA pol gamma, the Tyr955 amino acid plays a critical role in catalysis and high fidelity DNA synthesis. 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG) is one of the most common oxidative lesions in DNA and can promote transversion mutations. Mitochondria are thought to be a major source of endogenous reactive oxygen species that can react with dG to form 8-oxo-dG as one of the more common products. DNA polymerases can mitigate mutagenesis by 8-oxo-dG through allosteric interactions from amino acid side chains, which limit the anti-conformation of the 8-oxo-dG template base during translesion DNA synthesis. We demonstrated that the Y955C pol gamma displays relaxed discrimination when either incorporating 8-oxo-dGTP or translesion synthesis opposite 8-oxo-dG. Molecular modeling and biochemical analysis sug